18-ß-glycyrrhetinic acid-loaded polymeric nanoparticles attenuate cigarette smoke-induced markers of impaired antiviral response in vitro.

Tobacco smoking is a leading cause of preventable mortality, and it is the major contributor to diseases such as COPD and lung cancer. Cigarette smoke compromises the pulmonary antiviral immune response, increasing susceptibility to viral infections. There is currently no therapy that specifically addresses the problem of impaired antiviral response in cigarette smokers and COPD patients, highlighting the necessity to develop novel treatment strategies. 18-ß-glycyrrhetinic acid (18-ß-gly) is a phytoceutical derived from licorice with promising anti-inflammatory, antioxidant, and antiviral activities whose clinical application is hampered by poor solubility. This study explores the therapeutic potential of an advanced drug delivery system encapsulating 18-ß-gly in poly lactic-co-glycolic acid (PLGA) nanoparticles in addressing the impaired antiviral immunity observed in smokers and COPD patients. Exposure of BCi-NS1.1 human bronchial epithelial cells to cigarette smoke extract (CSE) resulted in reduced expression of critical antiviral chemokines (IP-10, I-TAC, MIP-1α/1ß), mimicking what happens in smokers and COPD patients. Treatment with 18-ß-gly-PLGA nanoparticles partially restored the expression of these chemokines, demonstrating promising therapeutic impact. The nanoparticles increased IP-10, I-TAC, and MIP-1α/1ß levels, exhibiting potential in attenuating the negative effects of cigarette smoke on the antiviral response. This study provides a novel approach to address the impaired antiviral immune response in vulnerable populations, offering a foundation for further investigations and potential therapeutic interventions. Further studies, including a comprehensive in vitro characterization and in vivo testing, are warranted to validate the therapeutic efficacy of 18-ß-gly-PLGA nanoparticles in respiratory disorders associated with compromised antiviral immunity.

18ß-Glycyrrhetinic acid protects against deoxynivalenol-induced liver injury via modulating ferritinophagy and mitochondrial quality control.

Deoxynivalenol (DON), a widespread mycotoxin, represents a substantial public health hazard due to its propensity to contaminate agricultural produce, leading to both acute and chronic health issues in humans and animals upon consumption. The role of ferroptosis in DON-induced hepatic damage remains largely unexplored. This study investigates the impact of 18ß-glycyrrhetinic acid (GA), a prominent constituent of glycyrrhiza, on DON hepatotoxicity and elucidates the underlying mechanisms. Our results indicate that GA effectively attenuates liver injury inflicted by DON. This was achieved by inhibiting nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, as well as by adjusting mitochondrial quality control (MQC). Specifically, GA curtails ferritinophagy by diminishing NCOA4 expression without affecting the autophagic flux. At a molecular level, GA binds to and stabilizes programmed cell death protein 4 (PDCD4), thereby inhibiting its ubiquitination and subsequent degradation. This stabilization of PDCD4 leads to the downregulation of NCOA4 via the JNK-Jun-NCOA4 axis. Knockdown of PDCD4 weakened GA's protective action against DON exposure. Furthermore, GA improved mitochondrial function and limited excessive mitophagy and mitochondrial division induced by DON. Disrupting GA's modulation of MQC nullified its anti-ferroptosis effects. Overall, GA offers protection against DON-induced ferroptosis by blocking ferritinophagy and managing MQC. ENVIRONMENTAL IMPLICATION: Food contamination from mycotoxins, is a problem for agricultural and food industries worldwide. Deoxynivalenol (DON), the most common mycotoxins in cereal commodities. A survey in 2023 showed that the positivity rate for DON contamination in food reached more than 70% globally. DON can damage the health of humans whether exposed to high doses for short periods of time or low doses for long periods of time. We have discovered 18ß-Glycyrrhetinic acid (GA), a prominent constituent of glycyrrhiza. Liver damage caused by low-dose DON can be successfully treated with GA. This study will support the means of DON control, including antidotes.

Identification of 18ß-glycyrrhetinic acid as an AGT inhibitor against LPS-induced myocardial dysfunction via high throughput screening.

Sepsis induced myocardial dysfunction (SIMD) is a serious complication of sepsis. There is increasing evidence that the renin-angiotensin system (RAS) is activated in SIMD. Angiotensinogen (AGT) is a precursor of the RAS, and the inhibition of AGT may have significant cardiovascular benefits. But until now, there have been no reports of small molecule drugs targeting AGT. In this study, we designed a promoter-luciferase based system to screen for novel AGT inhibitors to alleviate SIMD. As a result of high-throughput screening, a total of 5 compounds from 351 medicinal herb-derived natural compounds were found inhibiting AGT. 18ß-glycyrrhetinic acid (18ßGA) was further identified as a potent suppressor of AGT. In vitro experiments, 18ßGA could inhibit the secretion of AGT by HepG2 cells and alleviate the elevated level of mitochondrial oxidative stress in cardiomyocytes co-cultured with HepG2 supernatants. In vivo, 18ßGA prolonged the survival rate of SIMD mice, enhanced cardiac function, and inhibited the damage of mitochondrial function and inflammation. In addition, the results showed that 18ßGA may reduce AGT transcription by downregulating hepatocyte nuclear factor 4 (HNF4) and that further alleviated SIMD. In conclusion, we provided a more efficient screening strategy for AGT inhibitors and expanded the novel role of 18ßGA as a promising lead compound in rescuing cardiovascular disease associated with RAS overactivation.

Anti-psoriasis effect of 18ß-glycyrrhetinic acid by breaking CCL20/CCR6 axis through its vital active group targeting GUSB/ATF2 signaling.

BACKGROUND: Psoriasis is an immune-mediated chronic inflammatory skin disease. Current research suggests that the long-term persistence and recurrence of psoriasis are closely related to the feedback loop formed between keratinocytes and immune cells, especially in Th 17 or DC cells expressing CCR6. CCL20 is the ligand of CCR6. Therefore, drugs that block the expression of CCL20 or CCR6 may have a certain therapeutic effect on psoriasis. Glycyrrhetinic acid (GA) is the main active ingredient of the plant drug licorice and is often used to treat autoimmune diseases, including psoriasis. However, its mechanism of action is still unclear. METHODS: Psoriasis like skin lesion model was established by continuously applying imiquimod on the back skin of normal mice and CCR6-/- mice for 7 days. The therapeutic and preventive effects of glycyrrhetinic acid (GA) on the model were observed and compared. The severity of skin injury is estimated through clinical PASI scores and histopathological examination. qRT-PCR and multiple cytoline assay were explored to detect the expression levels of cytokines in animal dorsal skin lesions and keratinocyte line HaCaT cells, respectively. The dermis and epidermis of the mouse back were separated for the detection of CCL20 expression. Transcription factor assay was applied to screen, and luciferase activity assay to validate transcription factors regulated by GA. Technology of surface plasmon laser resonance with LC-MS (SPR-MS), molecular docking, and enzyme activity assay were used to identified the target proteins for GA. Finally, we synthesized different derivatives of 18beta-GA and compared their effects, as well as glycyrrhetinic acid (GL), on the skin lesion of imiquimod-induced mice to evaluate the active groups of 18beta-GA. RESULTS: 18ß-glycyrrhetinic acid (GA) improved IMQ-induced psoriatic lesions, and could specifically reduce the chemokine CCL20 level of the epidermis in lesion area, especially in therapeutic administration manner. The process was mainly regulated by transcription factor ATF2 in the keratinocytes. In addition, GUSB was identified as the primary target of 18ßGA. Our findings indicated that the subject on molecular target research of glycyrrhizin should be glycyrrhetinic acid (GA) instead of glycyrrhizic acid (GL), because GL showed little activity in vitro or in vivo. Apart from that, α, ß, -unsaturated carbonyl in C11/12 positions was crucial or unchangeable to its activity of 18ßGA, while proper modification of C3 or C30 position of 18ßGA may vastly increase its activity. CONCLUSION: Our research indicates that 18ßGA exerted its anti-psoriasis effect mainly by suppressing ATF2 and downstream molecule CCL20 predominately through α, ß, -unsaturated carbonyl at C11/12 position binding to GUSB in the keratinocytes, and then broke the feedback loop between keratinocytes and CCR6-expressing immune cells. GA has more advantages than GL in the external treatment of psoriasis. A highlight of this study is to investigate the influence of special active groups on the pharmacological action of a natural product, inspired by the molecular docking result.

18ß-Glycyrrhetinic acid exerts cardioprotective effects against BPA-induced cardiotoxicity through antiapoptotic and antioxidant mechanisms.

Bisphenol A (BPA) is a synthetic environmental pollutant widely used in industry, as well as is an endocrine disrupting chemicals and has a toxic effects on heart tissue. The aim of this study is to reveal the cardioprotective effects of 18ß-glycyrretinic acid (GA) against BPA-induced cardiotoxicity in rats. In this study, 40 male rats were used and five different groups (each group includes eight rats) were formed. The rats were applied BPA (250 mg/kg b.w.) alone or with GA (50 and 100 mg/kg b.w.) for 14 days. Rats were killed on Day 15 and heart tissues were taken for analysis. GA treatment decreased serum lactate dehydrogenase and creatine kinase MB levels, reducing BPA-induced heart damage. GA treatment showed ameliorative effects against lipid peroxidation and oxidative stress caused by BPA by increasing the antioxidant enzyme activities (glutathione peroxidase, superoxide dismutase, and catalase) and GSH level of the heart tissue and decreasing the MDA level. In addition, GA showed antiapoptotic effect by increasing Bcl-2, procaspase-3, and -9 protein expression levels and decreasing Bax, cytochrome c, and P53 protein levels in heart tissue. As a result, it was found that GA has cardioprotective effects on heart tissue by exhibiting antioxidant and antiapoptotic effects against heart damage caused by BPA, an environmental pollutant. Thus, it was supported that GA could be a potential cardioprotective agent.

Ursodeoxycholic acid and 18ß-glycyrrhetinic acid alleviate ethinylestradiol-induced cholestasis via downregulating RORγt and CXCR3 signaling pathway in iNKT cells.

Estrogen-induced intrahepatic cholestasis (IHC) is a mild but potentially serious risk and urges for new therapeutic targets and effective treatment. Our previous study demonstrated that RORγt and CXCR3 signaling pathway of invariant natural killer T (iNKT) 17 cells play pathogenic roles in 17α-ethinylestradiol (EE)-induced IHC. Ursodeoxycholic acid (UDCA) and 18ß-glycyrrhetinic acid (GA) present a protective effect on IHC partially due to their immunomodulatory properties. Hence in present study, we aim to investigate the effectiveness of UDCA and 18ß-GA in vitro and verify the accessibility of the above targets. Biochemical index measurement indicated that UDCA and 18ß-GA presented efficacy to alleviate EE-induced cholestatic cytotoxicity. Both UDCA and 18ß-GA exhibited suppression on the CXCL9/10-CXCR3 axis, and significantly restrained the expression of RORγt in vitro. In conclusion, our observations provide new therapeutic targets of UDCA and 18ß-GA, and 18ß-GA as an alternative treatment for EE-induced cholestasis.

Licorice metabolite 18ß-glycyrrhetinic acid activates G protein-gated inwardly rectifying K+ channels.

BACKGROUND AND PURPOSE: Licorice (liquorice) is a common food additive and is used in Chinese medicine. Excess licorice intake can induce atrial fibrillation. Patients with atrial fibrillation possess constitutively activated G protein-gated inwardly rectifying K+ (GIRK) channels. Whether licorice affects GIRK channel activity is unknown. We aimed to clarify the effects of licorice ingredients on GIRK current and the mechanism of action. EXPERIMENTAL APPROACH: A major component of licorice, glycyrrhizic acid (GA), and its metabolite, 18ß-glycyrrhetinic acid (18ß-GA), were tested. We performed electrophysiological recordings in Xenopus oocytes to examine the effects of GA and 18ß-GA on various GIRK subunits (Kir 3.1-Kir 3.4), mutagenesis analyses to identify the crucial residues for drug action and motion analysis in cultured rat atrial myocytes to clarify effects of 18ß-GA on atrial functions. KEY RESULTS: GA inhibited Kir 3.1-containing channels, while 18ß-GA activated all Kir 3.x subunits. A pore helix residue Phe137 in Kir 3.1 was critical for GA-mediated inhibition, and the corresponding Ser148 in Kir 3.2 was critical for 18ß-GA-mediated activation. 18ß-GA activated GIRK channel in a Gßγ -independent manner, whereas phosphatidylinositol 4,5-bisphosphate (PIP2 ) was essential for activation. Glu236 located at the cytoplasmic pore of Kir 3.2 appeared to be important to interactions with 18ß-GA. In rat atrial myocytes, 18ß-GA suppressed spontaneous beating via activation of GIRK channels. CONCLUSION AND IMPLICATIONS: GA acts as a novel GIRK inhibitor, and 18ß-GA acts as a novel GIRK activator. 18ß-GA alters atrial function via activation of GIRK channels. This study elucidates the pharmacological activity of licorice ingredients and provides information for drug design.

Design and synthesis of novel anti-multidrug-resistant staphylococcus aureus derivatives of glycyrrhetinic acid by blocking arginine biosynthesis, metabolic and H2S biogenesis.

With the soaring number of multidrug-resistant bacteria, it is imperative to develop novel efficient antibacterial agents and discovery new antibacterial pathways. Herein, we designed and synthesized a series of structurally novel glycyrrhetinic acid (GA) derivatives against multidrug-resistant Staphylococcus aureus (MRSA). The in vitro antibacterial activity of these compounds was evaluated using the microbroth dilution method, agar plate coating experiments and real-time growth curves, respectively. Most of the target derivatives showed moderate antibacterial activity against Staphylococcus aureus (S. aureus) and MRSA (MIC = 3.125-25 µM), but inactivity against Escherichia coli (E. Coli) and Pseudomonas aeruginosa (P. aeruginosa) (MIC > 200 µM). Among them, compound 11 had the strongest antibacterial activity against MRSA, with an MIC value of 3.125 µM, which was 32 times and 64 times than the first-line antibiotics penicillin and norfloxacin, respectively. Additionally, transcriptomic (RNA-seq) and quantitative polymerase chain reaction (qPCR) analysis revealed that the antibacterial mechanism of compound 11 was through blocking the arginine biosynthesis and metabolic and the H2S biogenesis. Importantly, compound 11 was confirmed to have good biocompatibility through the in vitro hemolysis tests, cytotoxicity assays and the in vivo quail chicken chorioallantoic membrane (qCAM) experiments. Current study provided new potential antibacterial candidates from glycyrrhetinic acid derivatives for clinical treatment of MRSA infections.

18ß-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling.

Endoplasmic reticulum stress (ERS)-induced inflammation participates in the occurrence of pulmonary arterial hypertension (PAH) by promoting pulmonary vascular remodeling, which involved in the activation of PERK/eIF2α/NF-κB signaling pathway. 18ß-Glycyrrhetinic acid (18ß-GA) has been found efficacious for attenuating PAH through its anti-remodeling effects in our previous research and it remains unclear whether 18ß-GA has an effect on the remodeling caused by ERS-induced inflammation. In this study, we made observations in monocrotaline-induced PAH rats and found improvement of hemodynamic and histopathological parameters, decreases in the right ventricular hypertrophy index, and alleviation of pulmonary vascular remodeling after 18ß-GA administration in vivo. Moreover, 18ß-GA could significantly inhibit the proliferation and DNA synthesis of human pulmonary arterial smooth muscle cells (HPASMCs) induced by platelet-derived growth factor BB. At the cellular and molecular levels, we found that 18ß-GA could significantly reduce the accumulation of misfolded protein in rat lung tissue, inhibit ERS activation, reduce the expression of GRP78, p-PERK, p-eIF2α, and p-NF-κB p65, and increase IκB protein expression. 18ß-GA could inhibit the migration of NF-κB into the nucleus, reduce the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatant of HPASMCs, and reduce GRP78, p-PERK, p-eIF2α, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression, increase IκB protein expression in HPASMCs. According to what we observed, this study indicated that 18ß-GA could treat PAH, which is related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.

18ß-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells.

BACKGROUND: Gastric carcinoma (GC) is a common gastrointestinal malignancy worldwide. Based on the cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. AIM: To explore the mechanism by which 18ß-glycyrrhetinic acid (18ß-GRA) regulates mitochondrial ribosomal protein L35 (MRPL35) related signal proteins to inhibit the proliferation of GC cells. METHODS: Cell counting kit-8 assay was used to detect the effects of 18ß-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823. The apoptosis and cell cycle were assessed by flow cytometry. Cell invasion and migration were evaluated by Transwell assay, and cell scratch test was used to detect cell migration. Furthermore, a tumor model was established by hypodermic injection of 2.5 × 106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18ß-GRA on GC cell proliferation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect MRPL35 expression in the engrafted tumors in mice. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry to screen for differentially expressed proteins (DEPs) extracted from GC cells and control cells after 18ß-GRA intervention. A detailed bioinformatics analysis of these DEPs was performed, including Gene Ontology annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and so on. Moreover, STRING database (https://string-db.org/) was used to predict protein-protein interaction (PPI) relationships and Western blot was used to detect the expression of proteins of interest in GC cells. RESULTS: The results indicated that 18ß-GRA could inhibit the proliferation of GC cells in a dose- and time-dependent manner. It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase. The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose, and the migration and invasiveness of GC cells were inhibited. The results of animal experiments showed that 18ß-GRA could inhibit tumor formation in BALB/c nude mice, and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice. Using TMT technology, 609 DEPs, among which 335 were up-regulated and 274 were down-regulated, were identified in 18ß-GRA intervention compared with control. We found that the intervention of 18ß-GRA in GC cells involved many important biological processes and signaling pathways, such as cellular processes, biological regulation, and TP53 signaling pathway. Notably, after the drug intervention, MRPL35 expression was significantly down-regulated (P = 0.000247), TP53 expression was up-regulated (P = 0.02676), and BCL2L1 was down-regulated (P = 0.01699). Combined with the Retrieval of Interacting Genes/Proteins database, we analyzed the relationship between MRPL35, TP53, and BCL2L1 signaling proteins, and we found that COPS5, BAX, and BAD proteins can form a PPI network with MRPL35, TP53, and BCL2L1. Western blot analysis confirmed the intervention effect of 18ß-GRA on GC cells, MRPL35, TP53, and BCL2L1 showed dose-dependent up/down-regulation, and the expression of COPS5, BAX, and BAD also increased/decreased with the change of 18ß-GRA concentration. CONCLUSION: 18ß-GRA can inhibit the proliferation of GC cells by regulating MRPL35, COPS5, TP53, BCL2L1, BAX, and BAD.

Physiology-Based Pharmacokinetic Study on 18ß-Glycyrrhetic Acid Mono-Glucuronide (GAMG) Prior to Glycyrrhizin in Rats.

To understand that 18ß-Glycyrrhetic acid 3-O-mono-ß-D-glucuronide (GAMG) showed better pharmacological activity and drug-like properties than 18ß-Glycyrrhizin (GL); a rapid and sensitive HPLC-MS/MS method was established for the simultaneous determination of GAMG and its metabolite 18ß-Glycyrrhetinic acid (GA) in rat plasma and tissues after oral administration of GAMG or GL. This analytical method was validated by linearity, LLOQ, specificity, recovery rate, matrix effect, etc. After oral administration, GAMG exhibited excellent Cmax (2377.57 ng/mL), Tmax (5 min) and AUC0-T (6625.54 mg/L*h), which was much higher than the Cmax (346.03 ng/mL), Tmax (2.00 h) and AUC0-T (459.32 mg/L*h) of GL. Moreover, GAMG had wider and higher tissue distribution in the kidney, spleen, live, lung, brain, etc. These results indicated that oral GAMG can be rapidly and efficiently absorbed and be widely distributed in tissues to exert stronger and multiple pharmacological activities. This provided a physiological basis for guiding the pharmacodynamic study and clinical applications of GAMG.

18ß-Glycyrrhetinic acid monoglucuronide (GAMG) alleviates single-walled carbon nanotubes (SWCNT)-induced lung inflammation and fibrosis in mice through PI3K/AKT/NF-κB signaling pathway.

Carbon nanotubes (CNTs) have become far and wide used in a number of technical and merchant applications as a result of substantial advances in nanotechnology, therein single-walled carbon nanotubes (SWCNT) are one of the most promising nanoparticles. Inhaling CNTs has been linked to a variety of health problems, including lung fibrosis. Glycyrrhetinic acid 3-O-mono-ß-D-glucuronide (GAMG), a natural sweetener, has anti-inflammatory and antioxidant capacities. The purpose of this study was to evaluate the potential for GAMG to alleviate SWCNT-induced lung inflammation and fibrosis. During days 3-28 after SWCNT intratracheal administration, we observed a remarkable increase of IL-1ß, IL-6 and TNF-α in bronchoalveolar lavage fluid (BALF) on day 3 and collagen deposition on day 28. GAMG treatment remarkably ameliorated SWCNT-induced pulmonary fibrosis and attenuated SWCNT-induced inflammation and collagen deposition, and suppressed the activation of PI3K/AKT/NF-κB signaling pathway in the lungs. Therefore, GAMG has a therapeutic potential for the treatment of SWCNT-induced pulmonary fibrosis. Targeting PI3K/AKT/NF-κB signaling pathway may be a potential therapeutic approach to treat pulmonary fibrosis in mice with SWCNT.

Neuroprotective effects of 18ß-glycyrrhetinic acid against bisphenol A-induced neurotoxicity in rats: involvement of neuronal apoptosis, endoplasmic reticulum stress and JAK1/STAT1 signaling pathway.

The exposure to bisphenol A (BPA) is inevitable owing to its common use in the production of polycarbonate plastics. Studies to reduce side effects are gaining importance since BPA causes severe toxicities in important tissues such as testes, lungs, brain, liver and kidney. The current study was planned to study ameliorative effect of 18ß-glycyrrhetinic acid (18ß-GA) on BPA induced neurotoxicity. Fourty Wistar albino rats were divided into five equal groups as follows: I-Control group, II-18ß-GA group (100 mg/kg), III- BPA group (250 mg/kg), IV-250 mg/kg BPA + 50 mg/kg 18ß-GA group, V-250 mg/kg BPA + 100 mg/kg 18ß-GA group. BPA intoxication was associated with increased MDA level while reduced GSH concentration, activities of glutathione peroxidase, superoxide dismutase, and catalase. BPA supplementation caused apoptosis in the brain by up-regulating caspase-3 and Bax levels and down-regulating Bcl-2. BPA also caused endoplasmic reticulum (ER) stress by increasing mRNA transcript levels of PERK, IRE1, ATF-6 and GRP78. Additionally, it was observed that BPA administration activated JAK1/STAT1 signaling pathway and levels of TNF-α, NF-κB, p38 MAPK and JNK in the brain. However, co-treatment with 18ß-GA at a dose of 50 and 100 mg/kg considerably ameliorated oxidative stress, inflammation, apoptosis, ER stress and JAK1/STAT1 signaling pathway in brain tissue. Overall, the data of this study indicate that brain damage associated with BPA toxicity could be ameliorated by 18ß-GA administration.

Transcriptional profile changes after treatment of ischemia reperfusion injury-induced kidney fibrosis with 18ß-glycyrrhetinic acid.

INTRODUCTION: Chronic kidney disease (CKD) is characterized by renal fibrosis without effective therapy. 18ß-Glycyrrhetinic acid (GA) is reported to have detoxification and anti-inflammatory functions and promotes tissue repair. However, the role of GA in CKD remains unclear. In this study, we investigated whether GA has a potential therapeutic effect in kidney fibrosis. METHODS: A renal fibrosis mouse model was established by ischemia/reperfusion (I/R) injury via clamping unilateral left renal pedicle for 45 min; then, the mice were treated with vehicle or GA. Kidney tissues and blood samples were extracted 14 days after reperfusion and renal function, histopathological staining, quantitative PCR, and western blotting were performed. RNA-seq was performed to explore the changes in the transcriptional profile after GA treatment. RESULTS: Renal function, pathological and molecular analysis displayed that fibrosis was successfully induced in the I/R model. In the GA treatment group, the severity of fibrosis gradually reduced with the best effect seen at a concentration of 25 mg kg -1. A total of 970 differentially expressed genes were identified. Pathway enrichment showed that reduced activation and migration of inflammatory cells and decreased chemokine interaction in significant pathways. Protein-protein interaction networks were constructed and 15 hub genes were selected by degree rank, including chemokines, such as C3, Ccl6, Ccr2, Ptafr, Timp1, and Pf4. CONCLUSIONS: GA may alleviate renal fibrosis by inhibiting the inflammatory response. GA is a promising therapy that may perhaps be used in treating renal fibrosis and CKD.

Single-crystal-to-single-crystal phase transition of 18ß-glycyrrhetinic acid isopropyl ester.

Due to the destruction of the integrity of the parent crystal, single-crystal-to-single-crystal phase transition in organic compounds is still a relatively rare phenomenon. The phase transition in glycyrrhetinic acid isopropyl ester is triggered by temperature change. The increasing volume of the isopropyl substituent as a result of increasing temperature forces a remodelling of the structural motifs. These changes cause a single-crystal-to-single-crystal phase transition. The low-temperature form is isostructural with glycyrrhetinic acid methanol solvate, while the high-temperature phase is isostructural with the ethyl ester of this acid.

Nrf2-mediated liver protection by 18ß-glycyrrhetinic acid against pyrrolizidine alkaloid-induced toxicity through PI3K/Akt/GSK3ß pathway.

BACKGROUND: Misusage of pyrrolizidine alkaloid (PA)-containing plants or unaware intake of PA-contaminated foodstuffs causes thousands of PA poisoning cases in humans. PA intoxication is accompanied by oxidative stress and subsequent extensive hepatocellular damage. Our previous study has demonstrated that 18ß-glycyrrhetinic acid (GA), a bioactive constituent of liquorice, prevented PA-induced hepatotoxicity in rats, however the underlying mechanisms remain unclear. OBJECTIVE: This study aims to explore the mechanisms underlying the hepato-protective effect of GA in combating retrorsine (RTS, a representative toxic PA)-induced liver injury. METHODS: Histological and biochemical assessments were employed to evaluate the protective effect of GA on RTS-induced hepatotoxicity in rats. Sulforhodamine B assay, real-time PCR, western blotting, and immunostaining were used to explore the underlying mechanisms in human hepatocytes and rats. RESULTS: Our findings demonstrated that GA alleviated RTS-induced elevation of serum ALT and bilirubin levels, as well as hepatocytes necrosis and sinusoidal endothelial cells (SECs) damage in rats. GA also enhanced the activities and expressions of several antioxidant enzymes through upregulating nuclear factor-erythroid 2-related factor2 (Nrf2). Moreover, inhibition of Nrf2 blocked the hepatoprotective effect of GA against RTS intoxication. Mechanistically, GA increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and enhanced glycogen synthase kinase 3 beta (GSK3ß) inhibitory phosphorylation at serine 9, thus promoting the nuclear accumulation of Nrf2 and activating its downstream targets. CONCLUSION: This study for the first time demonstrated that GA exerted protective effects against RTS-induced liver injury by potentiating the Nrf2-mediated antioxidant system through PI3K/Akt/GSK3ß pathway. The findings indicated that GA may serve as a potential candidate drug for the treatment of PA intoxication.

18ß-Glycyrrhetinic acid altered the intestinal permeability in the human Caco-2 monolayer cell model.

PURPOSE: Glycyrrhizin (GL) and its metabolites 18α-glycyrrhetinic acid (18α-GA) and 18ß-glycyrrhetinic acid (18ß-GA) are used as traditional medicine and food sweeteners. As the major rout of their administration is oral way, therefore their impact on intestinal epithelial cells are investigated. METHODS: The effects of GL and its metabolites on cell viability using MTT assay, on cytotoxicity using LDH release, on integrity of intestinal epithelial cells by measuring the transepithelial electrical resistance (TEER) and Luciferase permeability tests, on the expression of tight junction proteins at mRNA and protein level by qPCR and western blot techniques, and ultimately on the rate of test compounds absorption via Caco-2 cells monolayer were investigated. RESULTS: MTT assay showed a concentration- and time-dependent decrease in metabolic activity of Caco-2 cells induced by GL, 18α-GA, and 18ß-GA, while only 18ß-GA increased the LDH leakage. The monolayer integrity of Caco-2 cells in TEER assay only was affected by 18ß-GA. The permeability of paracellular transport marker was increased by 18α-GA and 18ß-GA and not GL. In transport studies, only metabolites were able to cross from Caco-2 cells monolayer. qPCR analyses revealed that 18ß-GA upregulated the expression of claudin-1 and -4, occludin, junctional adhesion molecules and zonula occludens-1, while 18α-GA upregulated only claudin-4. The expression of claudin-4 at protein level was downregulated non-significantly at 50 µM concentration of 18ß-GA. CONCLUSION: Our results suggest that 18ß-GA may cause cellular damages at higher concentrations on gastrointestinal cells and requires a remarkable attention of the nutraceutical and pharmaceutical industries.

In vitro growth inhibitory effect of selected 18ß-glycyrrhetinic acid esters on Theileriaannulata.

Glycyrrhetinic acid (GA) is one of the important Pentacyclic Triterpenoids (PT) found in the roots of licorice. This study aimed to evaluate the in vitro growth inhibitory effect of 18ß-GA (18ß-Glycyrrhetinic acid) and C-30 esters against Theileria annulata, the causative agent of Tropical Bovine Theileriosis. C-30 esters of 18ß-GA were synthesized and their structures were elucidated using spectroscopy. The pharmacodynamic properties of 18ß-GA and its C-30 esters were predicted using DataWarrior and Swiss ADME tools. Cattle isolates of T. annulata schizont-infected bovine lymphoblastoid cells were cultured using standard conditions and the growth inhibitory effect of GA and its esters were evaluated using MTT assay. The isopropyl ester of 18ß-GA (GI50- 1.638 µM; R2- 0.818) showed improved anti-theileriosis efficacy than other 18ß-GA derivatives. The propyl (GI50 - 5.549 µM), ethyl (GI50 - 5.638 µM), and benzyl (GI50 - 7.431 µM) esters also showed considerable inhibitory effect. The GI50 value for 18ß-GA was recorded as 6.829 µM. This study throws light on the usefulness of 18ß-GA and its esters for the treatment of Tropical Bovine Theileriosis.

Novel 18ß-glycyrrhetinic acid derivatives as a Two-in-One agent with potent antimicrobial and anti-inflammatory activity.

18ß-glycyrrhetinic acid (GA) is a well-known natural compound of oleanane-type triterpene and is found possessing antimicrobial and anti-inflammatory properties. Nonetheless, its relatively low bioactivity restricts its potential in pharmaceutical applications. To maximize the potential use of this natural herbal compound as antimicrobial and anti-inflammatory agents, the rational modification of GA to enhance its pharmacological activity with low toxicity and to understand the mechanism of action is critically essential. We reported herein the design and synthesis of a series of new GA derivatives. The antimicrobial activities of these new compounds were evaluated by inhibition zone test and minimum inhibitory concentration (MIC) assay. In addition, the anti-inflammatory activity was evaluated by LPS induced BV2 cells inflammation model and 12-O-tetradecanoyl phorbol-13-acetate (TPA) induced ear inflammation mice model. It was found that the derivatives functionalized with a di-substituted phenyl group at the 2-position of GA generally displayed high antimicrobial activity against Gram-positive bacteria (MIC down to 2.5 µM) and potent anti-inflammatory effects (inhibition of NO production up to 55%, comparable to dexamethasone). The in vitro and in vivo results also showed that GA-O-02 and GA-O-06 exert their anti-inflammatory activities through downregulation of NO, pro-inflammatory cytokines and chemokines (IL-1ß, IL-6, IL-12, TNF-α, MCP-1 and MIP-1α) and upregulation of anti-inflammatory cytokines (IL-10). The anti-inflammatory mechanism may involve the inhibition of NF-κB, MAPKs and PI3K/Akt related inflammatory signaling pathways and activation of Nrf2/HO-1 signaling pathway. The results demonstrated that GA-O-02 and GA-O-06 possess great application potential as potent antimicrobial and anti-inflammatory agents.